The creation and mass production of biotech based drugs represents a large endeavor and a significant segment of our economy. Many older methods of synthesising drugs are still in use while new techniques are constantly being developed. In particular, the subset of drugs that are biologically derived is rapidly growing. These biologically derived drugs are typically manufactured by growing cells that have been genetically engineered to produce a protein or other molecule of interest. These cells generally require an environment in which all or at least most of the temperature, pH, dissolved oxygen, dissolved CO2, glucose, lactate, and amino acid levels are monitored and/or controlled. A bioreactor is a vessel that is often used to provide such a controlled growth environment. These vessels have evolved over many years and have been designed and tested to ensure that they can provide the conditions required. However, irrespective of whether or not the bioreactor vessel itself can meet the requirements, control systems and sensors are always needed and must be monitored appropriately in order to consistently and reproducibly provide the desired conditions. Additionally, it is often necessary to document this reproducibility in order to meet the standards set forth by the pharmaceutical industry or the United States Food and Drug Administration (FDA). In order to meet the requirements of the industry with regard to reproducibility, sterility, robustness, and reliability new technologies are being developed that more accurately and economically allow a drug manufacturer to monitor and control the cell's environmental parameters. The end goal is to manufacture the desired end-product more reliably and at reduced cost.
As mentioned, the pharmaceutical industry must conform to many of the FDA's requirements which are intended to guarantee safe and effective drugs. Compliance with FDA requirements not only determines many of the processes that are put in place when manufacturing pharmaceutical and biotech products, but also drives many cost models for drug companies. One area that exemplifies the impact of FDA regulations on manufacturing methods and costs is specifically those regulations that pertain to the electronic equipment used in the manufacture of pharmaceuticals.
Specifically, The FDA enforces 21 CFR Parts 210 and 211 at all pharmaceutical production sites. Specifically, this requires that automated testing equipment systems perform satisfactorily and be checked for correct calibration on a regular basis (see e.g., 21 CFR section 211.68). Similar requirements often apply outside the United States. For example, in the EEC, Other requirements such as Commission Directives 91/356/EEX, 2003/94/EC, and 91/412/EEC may be applicable.
Phase fluorimetry is a technique that can be used to provide sensor instruments (“phase fluorimeters”) able to monitor many of the critical parameters in a bioreactor. Phase fluorimetry typically utilizes a fluorescent material (also referred to as a fluorescent dye or fluorophore) which has an upper-state lifetime that is quenched by the presence of a target analyte. Herein a fluorescent material is defined as a material which emits light at a lower frequency after illumination (stimulation) by an optical source. When sinusoidally modulated light is used to illuminate the fluorescent material, the emitted light is smaller in amplitude but is also sinusoidally modulated and delayed in phase with respect to the illumination light. This relationship between the illumination light and emitted light is shown in FIG. 1. In this Figure, 1 is the excitation source sine wave and 2 is the emitted or fluorescent signal's sine wave.
The difference in phase, Φ, between the excitation and emission light is proportional to the concentration of the quenching analyte and can therefore be used as an indicator of the concentration of that analyte. This difference is referred to variously as a delay or a shift. The terms are interchangeable in this situation. The fluorescent material, or fluorophore, must be selected or developed to be quenched by the particular analyte under study. This quenching is commonly modeled using the Stem-Volmer equation:
                                          F            0                    F                =                              γ            +                                          k                q                            ⁡                              [                Q                ]                                              γ                                    (                  Equation          ⁢                                          ⁢          1                )            
This quantifies the fractional change in fluorescence when quenching is increased. In this equation, γ is the decay rate of the fluorescence without additional quenching, kq is the bimolecular quenching constant, which measures the likelihood and effectiveness of quenching events, and [Q] is the concentration of the fluorophore. Both γ and kq are functions of temperature. This equation can be re-written to give the change in the decay time of the fluorophore as a function of quencher concentration. When this is done, the Stem-Volmer equation becomes
                              τ                      τ            0                          =                              γ                          γ              +                                                k                  q                                ⁡                                  [                  Q                  ]                                                              =                      1                          1              +                                                τ                  0                                ⁢                                                      k                    q                                    ⁡                                      [                    Q                    ]                                                                                                          (                  Equation          ⁢                                          ⁢          2                )            
Note that the decay rate is defined as the inverse of the characteristic decay time, τ0. I.e., γ−1τ0 (Lackowicz, Principles of Fluorescence Spectroscopy, 3rd edition, Springer 2006).
A typical set of data from a phase fluorimeter and an oxygen-quenched fluorescent dye is shown in FIG. 2. These curves are generated by utilizing a phase fluorimeter to measure known concentrations of the analyte at different temperatures. The sensitivity of the phase response to temperature in the range from 5° C. to 45° C. in steps of 10° C. is apparent in FIG. 2. This temperature range covers the operating temperature of the majority of bioprocesses.
Once these curves are measured they can be used to find functional forms (using algebraic or numerical fits) for kq and τ0 as functions of temperature or other relevant parameter and thus calibrate the sensor. Once the calibration is known, the oxygen concentration can be measured using an instrument that incorporates a phase fluorimeter and has had the fluorophore and instrument characterized by production of such curves. Without calibration, minor variations in any of the relevant parameters can cause a difference in phase that will give an erroneous reading.
The Stern-Volmer equation is one of the simplest equations describing the variation of fluorescence as a function of environmental parameters (γ, temperature, quencher concentration). More complicated equations exist and are used as the basis for phase fluorimeters. Calibration is done in a similar manner—the parameters that can impact phase in the specific situation where the phase fluorimeter is used are measured and their impact is corrected.
However, a problem arises because it is not uncommon for the properties of any fluorophore to change after a period of use. The analyte sensitive fluorescent dye's properties will frequently change as a result of long term exposure to both the analyte under study as well as to ambient light. This change in properties usually results in a change in the phase delay vs. concentration curve which means that the original calibration data is no longer valid and the readings are to a greater or lesser extent incorrect. Additionally, if the phase fluorimeter itself exhibits drift or is otherwise not capable of accurately and precisely reading the phase delay the result is a limit to the accuracy and precision of the sensor. As discussed above, in the pharmaceutical and biotech manufacturing sector, sensor performance is regulated by the Federal Code of Regulations and specifically, 21 CFR Part 11. The performance of a device used in the manufacturing of drugs is required to be tested for accuracy and function (e.g.: validated) on a regular and periodic basis in order to ensure that the drug efficacy and quality has not changed.